Contrast medium for near infrared diagnosis

ABSTRACT

This invention relates to colloidal systems charged with polymethine dyes and having suitable photophysical and pharmacological properties, their use as a contrast medium in fluorescence and transillumination diagnostics in the near infrared spectral range, as well as methods for their production.

CROSS REFERENCE

[0001] This application is a divisional application under 37 CFR§1.53(b) of allowed Ser. No. 09/051,511, entitled “Contrast Medium ForNear Infrared Diagnosis” filed Apr. 9, 1998.

FIELD OF THE INVENTION

[0002] This invention relates to colloidal systems charged withpolymethine dyes, their use as a contrast medium in fluorescence andtransillumination diagnostics in the near infrared spectral range, aswell as methods for their production.

BACKGROUND OF THE INVENTION

[0003] As biological tissue has relatively high permeability forlong-wave light in the range of 700 to 1000 ⁻nm, a diagnostician cantherefore use a completely different method of tissue imaging inaddition to advanced medical imaging techniques such as X-raying,magnetic resonance tomography, or ultrasonic diagnosis.

[0004] Tissue-specific information can be obtained both by detecting thenon-absorbed portion of radiation by means of a transmission image, andby detecting fluorescence radiation emitted after exposing the tissue tolight in the near infrared range.

[0005] The main problem with the use of near infrared radiation is theextraordinarily wide scattering of light, which yields only a ratherblurred image of a clearly contoured object despite differentphotophysical properties of such an object and its environment. Thisproblem intensifies the greater the distance from the surface and may beconsidered the major limiting factor of both transillumination and thedetection of fluorescence radiation.

SUMMARY OF THE INVENTION

[0006] Suitable fluorescent dyes that accumulate in diseased tissue(particularly in tumours) and display a specific absorption and emissionbehaviour may contribute towards enhancing the distinction of healthyfrom diseased tissue. The change in the irradiated (scattered) lightcaused by absorbtion of the dye, or fluorescence induced by excitingradiation, is detected and provides the actual tissue-specificinformation.

[0007] Until now, photosensitizers designed for use in photodynamictherapy (PDT) (including porphyrins, chlorines, phthalocyanines,naphthalocyanines) have been used for localizing and visualizing tumours(Bonnett R. New photosensitizers for the photodynamic therapy oftumours. SPIE Vol. 2078, 1994). The classes of compounds listed hereshare the disadvantage that in the wavelength range of 600 to 1200 nmthey are either not absorbing at all or to a very little extent only.The photosensitizing effect of these dyes is disturbing for purelydiagnostic uses where no effects are desired. Furthermore,photostability of the dyes listed here often is quite low.

[0008] In contrast to this, the absorption and fluorescent behaviour ofdyes from the class of polymethines is characterized by high absorptioncoefficients in the range between 700 and 1000 nm and a sufficientfluorescent quantum yield. The photosensitizing effect of polymethinescan be neglected, and most of them are highly photostable.

[0009] A clear change in the pharmacokinetic properties of fluorescentdyes may be achieved by transforming them into colloidal systems. As aresult, a tissue or organ-specific, or location-specific accumulation ofthe fluorescent dyes formulated in this way may be achieved.

[0010] Furthermore, the dyes must be strongly hydrophilic to be appliedin an aqueous solution so that sufficient quantities of dye for imagingcan be introduced into the body using a water solution.

[0011] It is known that the transformation of dyes into colloidalsystems may increase the applicable dose.

[0012] It is an object of this invention to provide a contrast mediumthat accumulates to a considerable extent in the tissues to be examinedand can be detected in said tissues using near infrared radiation.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] This problem is solved according to the invention by providingcontrast media containing colloidal systems charged with dye, saidcolloids having sizes from 5 nm to 10 μm and containing a minimum of onedye that absorbs and/or fluoresces in a wavelength range from 600 to1200 nm. For a definition of colloids, see Hunnius, PhamnazeutischesWörterbuch, 6th edition, Berlin: de Gruyter 1986, pp. 589 f.; thisdescription, however, includes coarsely dispersed systems with particlesizes up to 10 μm in the concept of colloidal systems.

[0014] Surprisingly, it was found that colloidal systems charged withdye accumulate in local inflammation or tumour areas. This accumulationis excellently suited for visualizing tumours or inflammations using NIRdiagnosis.

[0015] Furthermore, it was surprisingly found that the contrast media ofthe invention are quickly degraded in specific cells, for example, inKupfer cells of the liver, which results in a rapid decrease of colloidconcentrations in the blood, while the colloid particles alreadyabsorbed by the target tissue are degraded at a much slower pace. Thisreduces the background noise caused by the concentration of contrastmedium in the blood, which improves the visualization of lesions and/ormakes them visible at an earlier stage.

[0016] This invention thus relates to colloid systems charged with dyeand containing colloidal particles of sizes between 5 nm and 10 μm withdye molecules integrated in, adhering to, or encapsuled by their wallsand/or cavities.

[0017] Suited are all systems in which the dyes are integrated incolloidal particles consisting of biodegradable, partially syntheticmaterials or materials identical with natural substances. The propertiesand methods of preparing suitable colloidal particles are described, forexample, in Boyett, J. B., and Davis, C. W. Injectable Emulsions andSuspensions. In: Liebermann, H. A., M. M. Rieger, and G. S. Banker, eds.Pharmaceutical Dosage Forms: Disperse Systems. Vol. 2. New York: MarcelDekker, 1988, 379-416.

[0018] Particularly suitable are systems in which the syntheticpolymeric material has been selected from the following group:Poly-ε-caprolactone, polylactic acid, polyglycolic acid, and mixedpolymers from polylactic and polyglycolic acid, polyhydroxy butyricacid, polyhydroxy valeric acid, and mixed polymers from polyhydroxybutyric acid and polyhydroxy valeric acid, polyamino acids, polyalkylcyanoacrylates, polyamides, polyacrylodextrane, polyacrylic starch,polyacrylosaccharide, polyacrylamide, polyester, poly(ortho)ester,polyphosphorenes, and copolymers of lactic acid and/or glycolic acid andpolyoxyethylene. Preferred natural or partially synthetic biodegradablepolymeric materials primarily include proteins such as albumins,collagen, gelatin, haemoglobin or fibrinogen and starches, dextranes,chitin and chitosan.

[0019] Particularly suitable are also amphiphilic substances that eitherencapsule the fluorescent dye in colloidal particles or form colloidalparticles together with the fluorescent dye. Preferred substances arephospholipids, fatty acids, fatty alcohols, cholesterol, esters orethers of fatty alcohols or fatty alcohols and fatty acids, sugarderivatives with fatty acids or polyoxyethylene, esters or ethers ofphospholipids, fatty acids, fatty alcohols with polyoxyethylene, bileacids, derivatives of sorbitan with polyoxyethylene or fatty acids orfatty alcohols as well as their combinations.

[0020] Preferred contrast media according to the invention containcolloids from proteins such as albumins, collagen, gelatin, haemoglobinor fibrinogen, or starches and starch derivatives, dextranes, chitin, orchitosan.

[0021] Other preferred contrast media according to the invention containcolloids from phospholipids, fatty acids, fatty alcohols, cholesterol,esters of fatty alcohols and fatty acids, ethers of fatty alcohols andfatty acids, sugar derivatives containing fatty acids orpolyoxyethylene, esters or ethers of phospholipids, fatty acids or fattyalcohols with polyoxyethylene, bile acids, derivatives of sorbitan withpolyoxyethylene, fatty acids or fatty alcohols, and combinations of saidsubstances.

[0022] Particularly preferred are such contrast media of the inventionthat contain polyesters of α-, β-, γ- or ε-hydroxycarboxylic acids,polyalkyl cyanoacrylates, polyamino acids, polyamides, polyacrylatedsaccharides or poly(ortho)esters in the form of colloids.

[0023] The dyes used for the colloidal near-infrared diagnostic agentare characterized by the fact that they absorb and fluoresce in awavelength range of 600 to 1200 nm, have absorption coefficients ofabout 100,000 1 mol⁻¹ cm⁻¹ and higher, and, inasmuch as fluorescence isdesirable, show fluorescence quantum yields of more than 5%.

[0024] The dyes used are of the class of polymethine dyes selected fromthe following group: cyanine, styryl, merocyanine, squaraine, and oxonoldyes.

[0025] Preferred are dyes of the class of cyanine dyes having maximumabsorption and fluorescence values between 700 and 1000 nm andextinction coefficients of about 140,000 1 mol⁻¹ cm⁻¹ and more, andcarrying one or several unsubstituted, branched or non-branched, acyclicor cyclic or, optionally, aromatic carbon-hydrogen residues and/orcontaining oxygen, sulfur, nitrogen.

[0026] The contrast media of the invention contain a cyanine, styryl,merocyanine, squaraine, or oxonol dye, or a mixture of said dyes.

[0027] Preferred are contrast media according to the invention thatcontain a dye from the class of cyanine or squaraine dyes.

[0028] Also preferred are contrast media according to the invention inwhich the dye carries one or several additional, branched ornon-branched, cyclic or polycyclic alkyl, alkenyl, polyalkenyl, alkinyl,polyalkinyl, aryl, alkylaryl or arylalkyl residue(s), each containing upto 60 carbon atoms, and optionally carrying additional halogen atoms,hydroxy, carboxy, aminocarbonyl, alkoxycarbonyl, amino, aldehyde, oxo,oxy, or alkoxy groups that contain up to 20 carbon atoms, and/or may beinterrupted and/or replaced by one or several heteroatoms of the O, N,S, or P series.

[0029] Particularly preferred are contrast media of the invention thatcontain at least one dye of the general formula I, II, or III

[0030] wherein

[0031] R¹, R², R³, R⁴, R⁷, R⁸, R⁹, and R¹⁰ are the same or different,and each of which independently represents a —COOE¹, —CONE¹E², —NHCOE¹,—NHCONHE¹, —NE¹E², —OE¹, —SO₃E¹, —SO₂E¹, —SO₂NE¹E², —E¹ residue, afluorine, chlorine, bromine, or iodine atom or a nitro group, or where5- to 6-membered rings are anellated to each pair of adjacent residuesR¹, R², R³, or R⁴, R⁷, R⁸, R⁹ or R¹⁰ respectively, depending on the Catoms located in between, said rings being either saturated orunsaturated or aromatic and optionally carrying additional —COOE¹,—CONE¹E², —NHCOE¹, —NHCONHE¹, —NE¹E², —OE¹, —SO₃E¹, —SO₂E¹, —SO₂NE¹E²residues,

[0032] wherein

[0033] E¹ and E² are same or different, each of which independentlyrepresents a hydrogen atom, a saturated or unsaturated, branched ornon-branched C₁-C₅₀ alkyl chain, and said chain or parts thereofoptionally forms a cyclic C₅-C₆, or a bicyclic C₁₀ unit interruptedand/or replaced by oxygen atoms, sulfur atoms, nitrogen atoms,carboxylic acid ester, carboxylic acid amide, urea, thiourea, carbamateor ether groupings, or represent a hydroxypolyoxyethylene ormethoxypolyoxyethylene chain or a branched or non-branched C₁-C₁₀ alkylchain substituted with 1 to 19 fluorine atoms,

[0034] R⁵ and R⁶ independently represent an —E¹ residue or a C₁-C₄sulfoalkyl chain,

[0035] where

[0036] E1 is as defined above

[0037] Q represents a

[0038] fragment

[0039] wherein

[0040] R¹¹ is a hydrogen, fluorine, chlorine, bromine, or iodine atom, a—NE¹E², —OE¹, or —E¹ residue or a nitro group,

[0041] R¹² represents a hydrogen atom or an —E¹ residue,

[0042] b is one of the numbers 0, 2 or 3,

[0043] where

[0044] E¹ and E² are as defined above,

[0045] X and Y are same or different, each of which independentlyrepresents O, S, —CH═CH—, or a

[0046] fragment,

[0047] wherein

[0048] R¹³ and R¹⁴ independently represent hydrogen, a saturated orunsaturated, branched or non-branched C₁-C₁₅ alkyl chain, and where theresidues R¹³ and R¹⁴ may be interconnected by forming a 5- or 6-memberedring, said members being interrupted and/or replaced by oxygen atomsand/or hydroxy groups, alkoxy groups containing up to 6 carbon atoms,carboxylic acid ester and/or carboxylic acid amide units,

[0049] or their carboxylic acid and/or sulfonyl salts withphysiologically tolerable inorganic or organic cations.

[0050] Sodium, potassium, calcium, magnesium, gadolinium, and lysine,glutamine and methyl glutamine may occur as cations.

[0051] Dyes with a positive total charge are preferably iodides,bromides, or perchlorates.

[0052] The contrast media according to the invention may furthermorecontain a minimum of two dyes having different photophysical and/orpharmacological properties.

[0053] The contrast media according to the invention may furthermorecontain adjuvants, substrates, and diluents common in galenics.

[0054] The dyes are prepared using methods known from the relevantliterature as, for example, Hamer, F. M. The Cyanine Dyes and RelatedCompounds. New York: John Wiley and Sons, 1964; Bioconjugate Chem. 4(1993), 105-11; Anal. Biochem. 217 (1994), 197-204; Tetrahedron 45(1989), 4845-66; Anal. Chim. Acta 282 (1993) 633-641; Dyes Pigm. 21(1993), 227-234; EP 0 591 820 A1.

[0055] Another object of this invention is the use of the contrast mediaof the invention for in vivo fluorescence and absorption diagnosis inthe near infrared range.

[0056] In vivo diagnosis using the contrast media of the invention ispreferably carried out after intravenous administration of thesubstances by irradiating with monochrome light of a wavelength range of600 to 1200 nm and position-dependent detection of non-absorbedradiation and/or fluorescent radiation. A synthetic image is generatedbased on the data obtained.

[0057] The diagnostic agent of the invention is prepared in analogy withthe methods for the production of colloidal particles described in therelevant literature in which one or several dye(s) is/are added to thereaction mixture. The charging ratio can be varied by setting thereaction system to another dye concentration.

[0058] The preferred method is to dissolve the organic materialsdescribed above together with one or more dyes in one or several organicsolvent(s) that cannot be mixed with water, and to emulgate them afteroptionally adding another solvent, an emulsifying agent may optionallybe added to the emulsion. The colloidal system obtained can subsequentlybe filtered and stabilized by drying, e.g. by lyophilizing.

[0059] These contrast media are also manufactured according to methodsknown to an expert skilled in the art which may involve the use ofcommon adjuvants and/or diluents, and the like. This includesphysiologically tolerable electrolytes, buffers, detergents, emulgators,and substances used to adjust osmolality or to enhance the stability andsolubility of the contrast media, e.g. cyclodextrines. The sterility ofthe preparations during the manufacturing process and, above all, whenapplying them, is to be ensured by common pharmaceutical measures.

[0060] The following examples shall explain the invention in some moredetail.

EXAMPLE 1 Production of a Particle Suspension Containing1,1′,3,3,3′,3′-hexamethyl Indotricarbocyanine Iodide

[0061] 7.6 mg of hexamethyl indotricarbocyanine iodide and 0.2 g of acopolymer of lactic acid and glycolic acid having a molecular mass ofabout 15,000 g/mol are dissolved in 2.5 ml of methylene chloride. Thissolution is added under vigorous stirring to 20 ml of a 2% solution ofgelatin that has been autoclaved for 15 minutes at 121° C. The mixtureis kept agitated for another 45 minutes. The suspension thus obtained isfilled into 20 ml glass vessels in portions of 5 ml and frozen directlyusing liquid nitrogen. Subsequently, the frozen suspension islyophilized. After re-suspending a portion with 5 ml of a 0.9% solutionof common salt, the suspension contains about 10¹⁰ particles containinghexamethyl indotricarbocyanine iodide per ml with particle sizes fromabout 1 to 10 μm.

EXAMPLE 2 Application of the Preparation from Example 1

[0062] Fluoroscopic imaging of a nude mouse with a LS174T tumour afterapplying 200 μl of a poly-(1-lactide-glycolide) particle suspension(10¹⁰ particles /ml) with encapsuled hexamethyl indotricarbocyanineiodide (0.6 μmol/ml) using a CCD camera yielded images showingfluorescence mainly in the tumour tissue after 24 hours (FIG. 1).

1. A contrast medium for near infrared diagnosis that contains colloidalsystems charged with dye, characterized in that the colloidal particleshave a size from 5 nm to 10 μm, and that they contain at least one dyeabsorbing and/or fluorescing in the wavelength range from 600 to 1200nm.
 2. The contrast medium according to claim 1, characterized in thatthe colloids contain polyesters of α-, β-, γ-, or εe-hydroxycarboxylicacids, polyalkyl cyanoacrylate, polyamino acids, polyamides,polyacrylated saccharides, or poly(ortho)esters.
 3. The contrast mediumaccording to claim 1, characterized in that the colloids containproteins such as albumins, collagen, gelatin, haemoglobin or fibrinogen,or starches, dextranes, chitin or chitosan.
 4. The contrast mediumaccording to claim 1, characterized in that the colloids containphospholipids, fatty acids, fatty alcohols, cholesterol, esters of fattyalcohols and fatty acids, ethers of fatty alcohols, sugar derivativeswith fatty acids or polyoxyethylene, esters or ethers of phospholipids,fatty acids, or fatty alcohols with polyoxyethylene, bile acids,derivatives of sorbitan with polyoxyethylene, fatty acids, or fattyalcohols, as well as combinations of said substances.
 5. A contrastmedium according to at least one of claims 1 to 4, characterized in thatit contains as its dye component a cyanine, styryl, merocyanine,squaraine, an oxonol dye, or a mixture of said dyes.
 6. A contrastmedium according to at least one of claims 1 to 4, characterized in thatit contains at least one dye from the class of cyanine dyes or squarainedyes.
 7. The contrast medium according to claim 6, characterized in thatsaid dye carries one or several additional unbranched, branched, cyclicor polycyclic alkyl, alkenyl, polyalkenyl, alkinyl, polyalkinyl, aryl,alkylaryl, or arylalkyl residues, each containing up to 60 carbon atomswhich may optionally carry additional halogen atoms, hydroxy, carboxy,aminocarbonyl, alkoxycarbonyl, amino, aldehyde, oxo, oxy, or alkoxygroups containing up to 20 carbon atoms and/or optionally interruptedand/or replaced by one or several additional heteroatoms from the O, N,S, or P series.
 8. The contrast medium according to claim 7,characterized in that it contains at least one dye of the generalformula I, II, or III

wherein R¹, R², R³, R⁴, R⁷, R⁸, R⁹, and R¹⁰ are same or different, eachof which independently represents a —COOE¹, —CONE¹E², —NHCOE¹,—NHCONHE¹, —NE¹E². —OE¹, —SO₃E¹, —SO₂E¹, —SO₂NE¹E², —E¹ residue, afluorine, chlorine, bromine, or iodine atom or a nitro group, or where5- to 6-member rings are anellated to each pair of adjacent residues R¹,R², R³, or R⁴, and R⁷, R⁸, R⁹ or R¹⁰ respectively, depending on the Catoms located in between, said rings being either saturated orunsaturated or aromatic and optionally carrying additional —COOE¹,—CONE¹E², —NHCOE¹, —NHCONHE¹, —NE¹E², —OE¹, —SO₃E¹, —SO₂E¹, —SO₂NE¹E²residues, wherein E¹ and E² are same or different, each of whichindependently represents a hydrogen atom, a saturated or unsaturated,branched or non-branched C₁-C₅₀ alkyl chain, and said chain or partsthereof optionally form a cyclic C₅-C₆, or a bicyclic C₁₀ unitinterrupted and/or replaced by oxygen atoms, sulfur atoms, nitrogenatoms, carboxylic acid ester, carboxylic acid amide, urea, thiourea,carbamate, ether groupings, or represent a hydroxypolyoxyethylene ormethoxypolyoxyethylene chain or a branched or non-branched C₁-C₁₀ alkylchain substituted with 1 to 19 fluorine atoms, R⁵ and R⁶ independentlyrepresent an —E¹ residue or a C₁-C₄ sulfoalkyl chain, where E1 is asdefined above Q represents a

fragment wherein R¹¹ is a hydrogen, fluorine, chlorine, bromine, oriodine atom, a —NE¹E², —OE¹, or —E¹ residue or a nitro group, R¹²represents a hydrogen atom or an —E¹ residue, b is one of the integers0, 2 or 3, where E¹ and E² are as defined above, X and Y are same ordifferent, each of which independently represents O, S, —CH═CH—, or a

fragment, wherein R¹³ and R¹⁴ independently represent hydrogen, asaturated or unsaturated, branched or non-branched C₁-C₁₅ alkyl chain,and where the residues R¹³ and R¹⁴ may be interconnected by forming a 5-or 6-member ring, said members being interrupted and/or replaced byoxygen atoms and/or hydroxy groups, alkoxy groups containing up to 6carbon atoms, carboxylic acid ester and/or carboxylic acid amide units,or their carboxylic acid and/or sulfonyl salts with physiologicallytolerable inorganic or organic cations.
 9. Contrast medium according toat least one of claims 1 to 8, characterized in that it contains aminimum of two dyes with different photophysical and/or pharmacologicalproperties.
 10. Contrast medium according to at least one of claims 1 to9, together with the adjuvants, substrates, and diluents common ingalenics.
 11. Use of contrast media according to at least one of claims1 to 10 for in vivo fluoroscopy and absorption diagnosis in the nearinfrared range.